Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage λ

Chattopadhyay, S. ; Garcia-Mena, J. ; DeVito, J. ; Wolska, K. ; Das, A. (1995) Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage λ PNAS, 92 (9). pp. 4061-4065. ISSN 0027-8424

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Transcription of downstream genes in the early operons of phage λ requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of λ N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Item Type:Article
Source:Copyright of this article belongs to National Academy of Sciences.
ID Code:60249
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